PROJECT SUMMARY/ABSTRACT T cells belonging to the gd-lineage have been shown to serve unique and critical roles within the immune system. gd T cells are widely distributed throughout mucosal and epithelial cell-rich tissues and are an important early source of IL-17, which recruits granulocytes to sites of inflammation in response to pathogens. However, dysregulation of IL-17 production by gd T cells has also been linked to multiple pathological conditions including autoimmune disease and cancer progression. Unlike conventional ab T cells, gd T cells can acquire the ability to rapidly produce inflammatory cytokines within the thymus during development, prior to antigen exposure. The molecular and cellular events that specify and assign gd T cells to the IL-17 (gdT17) or interferon-g (IFNg; gdT1) effector fates during development remain to be fully elucidated. Recent evidence indicates that E proteins, in particular HEB, are critical mediators of this fate choice. HEB transcription factors are encoded by the Tcf12 gene locus, which gives rise to two proteins, HEBAlt and HEBCan, that differ in their structure, transcription initiation sites, and expression patterns. HEBAlt and HEBCan are expressed at partially overlapping times during gd T cell development, but their respective roles in functional programming of gd T cell precursors are unclear. We and others have identified a number of signaling pathways that contribute to gd T cell effector fate acquisition, including those that operate through T cell receptor (TCR), Notch, and cytokine receptors. It is our goal to understand how HEB activity is integrated with other determinants of gd T cell programming. Given our recent finding that HEB factors are required for the development of gdT17, but not gdT1 cells, we hypothesize that the final differentiation and effector function programming of gd T cells is directed by the interplay of specific HEB and E2A proteins with transcriptional regulators downstream of TCR, Notch, and cytokine receptor signaling. We will take advantage of a new in vitro model system that directs fate bifurcation between the gdT1 and gdT17 fates, as well as novel mouse models that we have generated, to elucidate the roles for E protein dimers containing HEBAlt, HEBCan, and/or E2A in determining gd T cell effector function, and to gain insight into the molecular basis for these events by assembling global gene regulatory networks. Our aims are: 1) to define the roles of HEBAlt, HEBCan, and E2A in intrathymic gd T cell differentiation by identifying specific target genes and binding partners in WT and mutant mice; and, 2) to evaluate the roles of HEB factors and their targets in human gd T cell development. Our proposal is built upon key observations derived from all Program Projects. The proposed experiments will continue to draw the complementary expertise in the areas of TCR signaling (Proj.1), mouse genetics (Proj.2), and chromatin remodeling (Proj.4). Our specific aims will not only lead to a better understanding of the mechanism of gd differentiation but also provide unique insights into the roles of specific E proteins in T cell development and function. Taken together, our joint experimental approaches and conceptual integration would not be possible without the combined expertise of all members of this Program.